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目的 :构建并在真核细胞中表达抗人重组碱性成纤维生长因子 (rh bFGF)人 鼠嵌合抗体。方法 :从分泌抗rh bFGF鼠单抗(mAb) 1F11的杂交瘤细胞中扩增、克隆VL、VH 基因 ,从质粒pMDHC和pMDLC中扩增、克隆CL、CH 基因 ,测序鉴定后插入真核表达载体 ,并转化二氢叶酸还原酶缺陷型中国仓鼠卵巢(CHO dhfr- )细胞进行表达。采用间接ELISA检测表达产物的人源性和其抗原结合活性。结果 :序列测定表明所克隆的抗体基因是功能性抗体的V区基因 ,在转化细胞的培养上清中可检测到抗rh bFGF嵌合抗体的表达。ELISA试验证实 ,该嵌合抗体具有人源C区 ,并具有鼠mAb 1F11的抗原结合活性。结论 :在真核细胞中成功地表达抗rh bFGF的人 鼠嵌合抗体 ,为其临床应用研究奠定了基础
OBJECTIVE: To construct and express chimeric antibodies against human recombinant human basic fibroblast growth factor (rh bFGF) in eukaryotic cells. Methods: The VL and VH genes were amplified from hybridoma cells secreting anti-rhbFGF monoclonal antibody (mAb) 1F11. The VL and VH genes were amplified and cloned from pMDHC and pMDLC. CL and CH genes were cloned and sequenced. Vector, and transformed into dihydrofolate reductase-deficient Chinese hamster ovary (CHO dhfr-) cells for expression. Indirect ELISA was used to detect the human origin and antigen-binding activity of the expressed product. Results: Sequence analysis showed that the cloned antibody gene was the V region gene of functional antibody, and the expression of anti-rh bFGF chimeric antibody was detected in the culture supernatant of the transformed cells. The ELISA test confirmed that the chimeric antibody has a human C region and has the antigen-binding activity of the murine mAb 1F11. CONCLUSIONS: The successful cloning of human chimeric antibodies against human rhbFGF in eukaryotic cells has laid the foundation for its clinical application